The long range goal of this project has been to understand how phorbol ester tumor promoters affect cell growth and differentiation. Since identification of the phorbol ester receptor as Protein Kinase C (PKC), we have focussed on the signal transduction pathways leading from activation of PKC to control of gene expression. Identification of multiple PKC isozymes and evidence for distinct roles for the isozymes has complicated elucidation of the signalling pathways. It is now essential to better understand how the individual isozymes are activated in order to know which ones will be activated under various physiological and pathological conditions. This will also facilitate design of drugs to specifically activate or inhibit individual isozymes. Toward this end, the next grant will focus on understanding the mechanisms for activation at a chemical and biophysical level. Aim I will test the hypothesis that specific physical states of the membrane are required for activation of PKC. This hypothesis arose from our finding that non- acidic micelles can activate PKC and that activation in long chain lipids requires unsaturation in the lipid acyl chains. PKC activity and membrane binding will be correlated with fluidity, phase structure, phase separation, and head group spacing. These features will be monitored with fluorescent probes, calorimetry, and 31P NMR spectroscopy. Membrane structure will be manipulated by changing lipid acyl chain composition and adding cholesterol and lipid active drugs. We will also ask whether the PKC cofactors Ca2+, diacylglycerol, phorbol esters contribute to activation in part by promoting formation of the appropriate physical state of the membrane. Aim II is to determine the structural basis for differential activation of PKC isozymes by cofactors. The unique high affinity Ca2+ site identified on PKCalpha and the phosphatidyl serine- dependent site on PKCbeta will be located, as will Mg2+, nucleotide, and peptide substrate sites. Interactions between the cofactors and substrates will be characterized by water proton relaxation NMR and EPR studies. Paramagnetic metals and 13C- and spin labeled lipid and PKC probes will be employed. In Aim III we will examine the effects of PKC phosphorylation on steps in the activation including cofactor and substrate binding, PKC conformation, and membrane binding. In Aim IV we will initiate attempts to selectively activate/inhibit individual PKC isozymes in cells based on what we have learned about their distinct activation mechanisms.